Multi-omics analysis reveals Yishen Qutong formula promotes ferroptosis in Lewis lung cancer associated with Keap1/Nrf2/Nqo1 pathway modulation

ETHNOPHARMACOLOGICAL RELEVANCE: Yishen Qutong Formula (YSQTF) is composed of six ingredients of Liuwei Dihuang, a classical traditional Chinese medicine fomula, plus Drynaria fortunei (Kunze) J. Sm., Bombyx Batryticatus, Hedyotis diffusa Willd, Scutellaria barbata D. Don. YSQTF has been proved effective in clinical practice of cancer pain. However, its role and mechanisms for anti-tumor activity remain unclear. AIM OF THE STUDY: The objective of this research is to identify the principal constituents, pivotal targets, and possible therapeutic mechanisms of YSQTF in the Lewis lung cancer treatment. MATERIALS AND METHODS: Widely targeted metabolomics was used to identify the serum components in YSQTF. Using network pharmacology, we identified potential YSQTF targets and conducted enrichment analysis on them. Subsequently, we investigated the regulatory effects of YSQTF on the Keap1/Nrf2 pathway and ferroptosis in lewis lung cancer cells through RT-qPCR, Western blot and single cell sequencing. We used untargeted metabolomics to evaluate the regulatory effect of YSQTF-containing serum on the metabolism of Lewis cells. Finally, we investigated the potential serum component regulating Keap1/Nrf2 pathway through molecular docking analysis. RESULTS: The study identified 39 compounds as YSQTF serum components and predicted 550 genes as their potential targets. In vitro, three concentrations of YSQTF (10%, 20%, and 30% serum) dose-dependently upregulated Keap1 while suppressing Nrf2 and Nqo1 in lewis cells. In vivo, three doses of YSQTF (2.21, 4.42 or 6.63 g/kg) exerted similar dose-dependent effects on these proteins in tumor tissues. These changes collectively promoted ferroptosis, as evidenced by elevated iron, MDA, 4-HNE, and ROS levels, alongside decreased GSH, GSSG, and GSH/GSSG ratios in both cellular and animal models. 3-hydroxycinnamic acid in YSQTF exhibited potential regulatory effects on Keap1. CONCLUSION: YSQTF promoted ferroptosis in Lewis lung cancer, which was associated with the Keap1/Nrf2/Nqo1 pathway. 3-hydroxycinnamic acid might be a serum component in YSQTF regulating the expression of Keap1.